The invention relates to methods of plant regeneration. Specifically, methods utilizing media containing apical dominance inhibitors to induce multiple bud and shoot formation are disclosed. More specifically, methods for improving the efficiency of production of transgenic cotton and soybean plants are disclosed.
The expanding field of biotechnology provides the tools for scientists to introduce important traits into a variety of plant species. New technologies promote the production of commercially viable transgenic crops and have a significant economic impact on the agricultural industry. These advancements enable creation of new crop germplasm containing desirable novel traits. Such traits include improvements in the nutritional quality, insect resistance, disease resistance, and yield of many crops. Cotton is the leading fiber crop worldwide and holds significant agronomic influence in a number of markets. Accordingly, much effort is continually directed toward the genetic engineering of this agronomically important crop species.
Crop improvement is achieved with greater ease and rapidity when breeders are able to grow plant tissues in culture in such a way that whole plants, or portions thereof, can be rapidly produced from relatively high proportion of explants and from the most genotypically significant germplasm. The breeding and genetic engineering of cotton, however, has taken place at a relatively slow rate because of the absence of tissue culture methods capable of regenerating organized tissues, such as whole plants or shoots from cotton explants at a sufficiently high frequency and in a commercially timely manner from high-quality or commercial-quality germplasm.
Genetic engineering of plants is essentially a two-step process: transformation and regeneration. First, plant cells are transformed, thereby introducing a nucleic add sequence that is typically integrated into the genome of the host cell. Second, a sexually competent plant is regenerated from the transformed cells or tissues. In the most common method, nonembryogenic tissue is genetically transformed, then induced under suitable culture conditions to form somatic embryogenic calli. The embryos resulting therefrom may then be germinated to form plants. The transformation and regeneration processes preferably are complementary such that the successfully transformed tissues are capable of developing into competent whole plants.
Current systems are very time-consuming and laborious and a high frequency of plants that are recovered are not fertile. Meristem-based transformation systems are advantageous in that they are faster, but the plants are chimeras and it is difficult to identify germline transformants. If multiple shoots could be produced from the transformed meristem cells, then improved selection schemes could be utilized and nonchimeric transformed plants could be produced. Typically, cytokinins are used to produce a multiple shoot response in plants. However, in cotton, cytokinins only produce a few shoots in many cultivars or kill the explants entirely.
The present invention provides a method of culturing cotton and soybean that produces multiple shoots in culture and allows for the recovery of fertile plants. This allows for more efficient transformation and regeneration of cotton and soybean.